This application is a 371 of PCT/ES98/00045, filed Feb. 26, 1998.
Physical Medicine
Pharmacy
Biomedical Research
Imaging Diagnostics
The biomedical applications of Magnetic Resonance (MR) have experienced an important development over the last few decades (Andrew, E. R., Byders, G., Griffiths, J., Iles, R. and Styles, P. Eds (1990) Clinical Magnetic Resonance Imaging and Spectroscopy. John Wiley and Sons. New York) Both Magnetic Resonance Spectroscopy (MRS) and Imaging (MRI) have been used for non-invasive studies on both animal and human pathological and physiological processes (Gillies, R. J. Ed. (1994) NMR in Physiology and Biomedicine, Academic Press, New York). Further, it has been possible to extend the applications of MR to study pathological and physiological processes at the cell level (Gillies, R. J., Gallons, J. P., McGovern, K. A., Scherrer, P. G., Lien, Y. H., Job, C. R., Chapa, F., Cerdan, S. and Dale, B. E. (1993) NMR in Biomedicine 6,95-104). Taken together, these advances have permitted to apply MR to study some fundamental aspects of Biology and Medicine, like cellular proliferation and differentiation, or tumoral transformation. These processes do occur with accompanying changes in intra and extracellular pH. Further, pH is a crucial physiological variable in other fundamental processes such as physical exercise and muscle fatigue, metabolic control, and hormonal message transduction (Roos, A. and Boron, W. F. (1981) Physiol. Rev 61, 296-696).
The intracellular proton concentration results from a balance between a number of factors including the intracellular proton production and consumption, proton uptake from and extrusion to the extracellular medium, and the intracellular proton buffering capacity (Molde, M., Cruz, F., Chapa, F., Cerdan, F. and Cerdan, S. (1995) Quart. Mag. Res. in Biol. Med. 2, 5-17). Considering that the latter is fairly constant under normal conditions, an increase in intracellular proton production may be balanced by either acid extrusion to or base uptake from the extracellular medium; whereas an increased intracellular base production could be compensated by the opposite mechanism. Thus, changes in extracellular pH normally reflect a change in intracellular pH, and measuring extracellular pH provides a useful mean to monitor any alteration in intracellular pH homeostasis.
Methods currently available for pH determination in biological samples include potentiometric methods (pH electrodes), radiometric techniques, optical methods and Magnetic Resonance (Henderson, R. M. and Graf, J. (1988). In pH Homeostasis: Mechanisms and Control (Hxc3xa4ussinger, D. Ed.) Academic Press, pg. 5-26). In principle, any of these procedures is able to measure exclusively extracellular pH, provided that a non-permeable molecular probe, be it radioactive, chromophoric, fluorescent, phosphorescent, or active in the case of MR, is used. However, among all these methods, only MR allows one to collect non-invasive images from the entire volume of optically opaque samples. For this reason, MR is the most suitable method for non-invasive determinations of extracellular pH in biological specimens. In this patent we describe the use and applications of a new series of indicator molecules, which allow determining the intracellular and extracellular pH, using the MRS and MRI methods.
Previous approaches have mainly used 31P MRS and chemical shift of the inorganic phosphate to measure intracellular pH in cellular suspensions, perfused organs, tissue samples, intact animals and even human beings (Moon, R. B. and Richards, J. H. (1973) J. Biol. Chem. 248,7276-7278). A variation of this method applied to cell suspensions has made possible 1D-31P MRS measurements of the extracellular pH exclusively, by using phosphonates non-permeable to the plasma membrane (Guillies, R. J., Liu, Z. and Bhujawalla, Z. (1994) Am. J. Physiol. 267, C195-C203). On the other hand, localized 31P MRS techniques to obtain 31P NMR spectra from a restricted spatial region (voxel) within an intact animal, and thus determine the average intracellular pH for that particular region, using Pi chemical shift, have been previously reported (Aue, W. P. (1986) Rev. Mag. Res. Med. 1, 21-72; Ordidge, R. J., Connelly, A. and Lohman, J. A. B. (1986) J. Mag. Res. 66, 283-294; Frahm, J., Bruhn, H., Gyngell, M. N. L., Merboldt, K. D., Hanike, W. and Sauter, R. (1989) Mag. Res. Med. 9, 79-93). Multivoxel imaging 31P MRS methods have also been developed. With these techniques, it is feasible to obtain 31P NMR spectra simultaneously from a collection of adjacent voxels that completely span the 3-D volume of the specimen, which provides tridimensional maps of pH distribution throughout the sample (Brown, T. R., Kincaid, B. M. and Ugurbil, K. (1982) Proc. Natl. Acad. Sci. U.S.A., 79, 3523-3526; Maudsley, A. A., Hilal, S. K., Perman, W. H. and Simon, H. E. (1983). J. Mag. Res. 66, 283-294; Vigneron, D. B., Nelson, S. J., Nat, R., Murphy-Boesch, J., Kelley, D. A. C., Kessler, H. B., Brown, T. R. and Taylor, J. S. (1990) Radiology 177:643-649; Shungu, D. C. and Glickson, J. D. (1993) Mag. Res. in Med. 30, 661-71; Shungu, D. C. and Glickson, J. D. (1994) Mag. Res. in Med. 32, 277-84).
In spite of this progress, the low sensitivity of the 31P nucleus poses a limiting factor for the application of 31P MRS to pH determination. Thus, 31P MRS methods typically require long acquisition times as well as large voxel size, in order to have a good signal to noise ratio. These two requirements greatly reduce the spatial and temporal resolution of pH determination by 31P MRS. Both limitations can be lessen by using either 1H or 19F, as these nuclei are inherently more sensitive than 31P in MR (Gadian, D. G. (1982) Nuclear Magnetic Resonance and its applications to living systems. Oxford University Press. Pg. 8). The increased sensibility of 1H (19F) MRS compared to 31P MRS would allow one to obtain spectra, or 1-D, 2-D or 3-D images, with a similar signal to noise ratio as those from 31P MRS, but 4(3), 16(12) or 64(43) times faster. In like manner, using 1H (19F) MRS it would be possible to obtain a significant reduction in voxel size, by comparison to that required in 31P MRS acquisition, while keeping the same signal to noise ratio. However, the presence of intrinsic metabolites with the right 1H resonance is rather exceptional (Yoshisaki, K., Seo, Y. and Nishikawa, H. (1981) Biochem. Biophys. Acta 678, 283-291), and there are no natural metabolites containing 16F. Consistently and in order to successfully implement the 1H or 16F MRS (or MRI) technique for intracellular pH determination, the use of extrinsic probes containing 1H (Rabenstein, D. L. and Isab, A. (1982) Anal. Biochem. 121, 423) or 16F (Deutsch, C., Taylor, J. S. and Wilson, D. F. (1982) Proc. Natl; Acad. Sci; U.S.A., 79, 7944) pH sensitive nuclei is a must.
Part of our team has recently reported the synthesis of a new series of indicators for intracellular pH, extracellular pH and cellular volume 1H MNR measurements in cellular suspensions (Gil, M. S., Cruz, F., Cerdan, S. and Ballesteros, P. (1992) Bioorg. Med. Chem. Lett. 2, 1117-1722; Gil, M. S., Zaderenko, P., Cruz, F., Cerdan, S. and Ballesteros, P. (1994) Bioorg. Med. Chem. 2, 305-14; Zaderenko, P., Gil, M. S., Ballesteros, P. and Cerdan, S. (1994) J. Org. Chem. 59, 6268-73). In this patent, we described some pharmacological and toxicological properties of these molecules, in view of their use as pH indicators in cell cultures and intact animals; and we also explain the procedures followed to obtain pH images from both model systems in vitro as well as from mice carrying RIF-1 tumors in vivo. The use of these new indicators, in conjunction with 1H MR techniques, such as chemical shift imaging (CSI) or spectroscopic imaging (SI) results in a considerable reduction in acquisition time and a significant resolution increase for pH measurements, when compared with previous methods based on 31P MR.